Human plexin B1 effector domain

Target ID:

PLXNB1

Aliases:

KIAA0407PLEXIN-B1PLXN5SEP

Deposition Date:

Monday 27th August 2007

Families:

GTPases

Related Diseases:

Cancer

Structure type:

apo

Download Coordinates:

2R2O

Authors:

Tong Y, Tempel W, Shen L, Arrowsmith CH, Edwards AM, Sundstrom M, Weigelt J, Bochkarev A, Park H

Site:

Toronto

ICM Datapack:

ICM Datapack

2R2O

tabs

Structure Details

General Description

The plexin family of transmembrane proteins are receptors for semaphorins, axon growth cone guidance molecules (Nakamura et al., 2000). Plexins were first discovered in axons and developing nervous system (Tamagnone et al., 1999) and later found to play important roles in developmental processes such as cardiovascular development and angiogenesis (Toyofuku et al., 2004), in epithelial cell invasive growth (Giordano et al., 2002), and in the immune system (Wong et al., 2003). The extracellular region of plexins shares significant homology with that of the hepatocyte growth factor receptor and semaphorins. The cytoplasmic region of plexins is also well conserved (Tamagnone et al., 1999) and shows sequence similarity to RASA1, a GTPase activating protein (GAP) for Ras family GTPase (Rohm et al., 2000). The RasGAP homology region of plexins is split into two subdomains with an insertion of ~200 amino acid residues that bind to Rho GTPases, RAC1, RND1, and RHOD (Vikis et al., 2000;Driessens et al., 2001;Hu et al., 2001). Plexins are the first documented example of transmembrane receptors that interact directly with small GTPases.

Plexin B1 (PLXNB1) is a RasGAP for RRAS (Oinuma et al., 2004) and the GAP activity is involved in integrin-mediated actin remodeling and cell collapse (Ito et al., 2006). Binding of RND1 is required for the GAP activity of PLXNB1 for RRAS (Oinuma et al., 2004), representing a novel regulatory mechanism of the RasGAP activity by a Rho GTPase. We have further defined the RND1-binding region of PLXNB1 that consists of ~120 amino acid residues (Tong et al., 2007) and forms a dimer in solution (Tong et al., 2005). Mutations in this domain are associated with prostate cancer (Wong et al., 2007).

Structural features

We solved the crystal structure of the RND1-binding domain (the effector domain) of PLXNB1. The effector domain has an unusual ubiquitin fold (Figure 1): a central α-helix is surrounded by a five-strand β-sheet. Different from a typical ubiquitin fold, Helix α2 is located on a loop linking α1 and β3. Two cancer-related mutations, T1795A and T1802A are located in this loop (Wong et al., 2007) (Figure 2). Helix α2 has the lowest B-factor of the whole structure due to multiple hydrogen bond interactions between the main chain amide of R1797 and the carbonyl groups of P1793 and L1794, between the side chain guanidine group of R1797 with Y1789 and V1792. At the dimer interface, the 17 residues between β4 and α3 (S1818-N1834) of one molecule form a short β-hairpin, interacting in an antiparallel fashion with the same β-hairpin from the second molecule. W1830, the known residue to be critical for dimerization (Tong et al., 2005), is found at the dimer interface. The side chains of W1830 residues from the dimer make Van der Waals contact. The dimer interface is further stabilized by the hydrogen bonds between the backbones of residues L1829, R1831, L1833. The Residues 1757- 1763 are very flexible in the structure and not visible in electronic density map. This dimer structure has an implication for understanding the function of PLXNB1.

Materials and Methods

PLXNB1

PDB:2R2O

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:Provided by Dr. M.Buck (Case Western Reserve University, Cleveland, Ohio)
Entry Clone Source:Dr. Buck
SGC Clone Accession:HPC060-C12 (This construct will not be distributed since the DNA template used is a gift from Dr. Buck)
Tag:mhhhhhhssgrenlyfq*g
Host:BL21-CodonPlus(DE3)-RIL

Construct

Prelude:
Sequence:mhhhhhhssgrenlyfqgDVEYRPLTLNALLAVGPGAGEAQGVPVKVLDCDTISQAKEKMLDQLYKGVPLTQRPDPRTLDVEWRSGVAGHLILSDEDVTSEVQGLWRRLNTLQHYKVPDGATVALVPCLTKHVLRENQ
Vector:pET28-mhl

Growth

Medium:Terrific Broth
Antibiotics:
Procedure:LEX Bubbling. The target protein was expressed in E. coli by inoculating 100 mL of overnight culture grown in Luria-Bertani medium into a 1.8 L of Terrific Broth medium in the presence of 50 μg/mL kanamycin and chloramphenicol at 37 °C. When OD600 was ~3.0, the temperature of the media was lowered to 15 °C and the culutre was induced with 1mM IPTG, and the cells were allowed to grow overnight before harvesting and flash frozen in liquid nitrogen and stored at -80 °C before use.

Purification

Procedure
The lysate was centrigued at 15,000 rpm for 45 minutes and the supernatant was mixed with 5 mL 50% Ni-NTA beads, and incubated at 4 °C for 1 hours. The beads were then washed using washing buffer and the proteins eluted using 8 mL elution buffer twice. The elutants were pooled and loaded onto supderdex-75 gel filtration column. Eluted fractions were pooled and concentrated using amicon centrifugal filter (m.w. cut-off 10,000 ). The purity of the proteins was higher than 95% judged by SDS-PAGE.

Extraction

Procedure
Frozen cells were thawed and suspended in 150 mL the binding buffer and supplemented with protease inhibitor cocktail (SIGMA Catalog # P8849), and 2 microL (Sigma Catalog # E1014, 250U/microL) benzonase, and lysed using sonicator at 100W for 5 minutes ( duty cycle: 10s on, 5s off)
Concentration:26.8mg/mL, in 50mM Tris pH 7.5 150mMNaCl 5mMDTT/MgCl2 buffer
Ligand
N/AMassSpec:Measured 15417.85, expected 15417.47SeMet labeled: measured 15511.67, expected 15513.36
Crystallization:RG05 optimizaiton: NaCl 2.6M 0.1M Tris pH 8.0 18% EG50mM Tris pH 7.5 150 mM NaCl 5 mM DTT/MgCl2
NMR Spectroscopy:
Data Collection:
Data Processing:

References

1. Nakamura,F., Kalb,R.G., and Strittmatter,S.M. (2000). Molecular basis of semaphorin-mediated axon guidance. J. Neurobiol. 44, 219-229.

2. Tamagnone,L., Artigiani,S., Chen,H., He,Z., Ming,G.I., Song,H., Chedotal,A., Winberg,M.L., Goodman,C.S., Poo,M., Tessier-Lavigne,M., and Comoglio,P.M. (1999). Plexins are a large family of receptors for transmembrane, secreted, and GPI-anchored semaphorins in vertebrates. Cell 99, 71-80.

3. Toyofuku,T., Zhang,H., Kumanogoh,A., Takegahara,N., Suto,F., Kamei,J., Aoki,K., Yabuki,M., Hori,M., Fujisawa,H., and Kikutani,H. (2004). Dual roles of Sema6D in cardiac morphogenesis through region-specific association of its receptor, Plexin-A1, with off-track and vascular endothelial growth factor receptor type 2. Genes Dev. 18, 435-447.

4. Giordano,S., Corso,S., Conrotto,P., Artigiani,S., Gilestro,G., Barberis,D., Tamagnone,L., and Comoglio,P.M. (2002). The semaphorin 4D receptor controls invasive growth by coupling with Met. Nat. Cell Biol. 4, 720-724.

5. Wong,A.W., Brickey,W.J., Taxman,D.J., van Deventer,H.W., Reed,W., Gao,J.X., Zheng,P., Liu,Y., Li,P., Blum,J.S., McKinnon,K.P., and Ting,J.P. (2003). CIITA-regulated plexin-A1 affects T-cell-dendritic cell interactions. Nat Immunol. 4, 891-898.

6. Rohm,B., Rahim,B., Kleiber,B., Hovatta,I., and Puschel,A.W. (2000). The semaphorin 3A receptor may directly regulate the activity of small GTPases. FEBS Lett. 486, 68-72.

7. Vikis,H.G., Li,W., He,Z., and Guan,K.L. (2000). The semaphorin receptor plexin-B1 specifically interacts with active Rac in a ligand-dependent manner. Proc. Natl. Acad. Sci. U. S. A. 97, 12457-12462.

8. Driessens,M.H., Hu,H., Nobes,C.D., Self,A., Jordens,I., Goodman,C.S., and Hall,A. (2001). Plexin-B semaphorin receptors interact directly with active Rac and regulate the actin cytoskeleton by activating Rho. Curr. Biol. 11, 339-344.

9. Hu,H., Marton,T.F., and Goodman,C.S. (2001). Plexin B mediates axon guidance in Drosophila by simultaneously inhibiting active Rac and enhancing RhoA signaling. Neuron 32, 39-51.

10. Oinuma,I., Ishikawa,Y., Katoh,H., and Negishi,M. (2004). The Semaphorin 4D receptor Plexin-B1 is a GTPase activating protein for R-Ras. Science 305, 862-865.

11. Ito,Y., Oinuma,I., Katoh,H., Kaibuchi,K., and Negishi,M. (2006). Sema4D/plexin-B1 activates GSK-3beta through R-Ras GAP activity, inducing growth cone collapse. EMBO Rep. 7, 704-709.

12. Tong,Y.F., Chugha,P., Hota,P.K., Alviani,R.S., Li,M., Tempel,W., Shen,L., Park,H.W., and Buck,M. (2007). Binding of Rac1, Rnd1 and RhoD to a novel Rho GTPase interaction motif destabilizes dimerization of the plexin-B1 Effector domain. J. Biol. Chem. 282, 37215-37224.

13. Tong,Y.F., Hughes,D., Placanica,L., and Buck,M. (2005). When monomers are preferred: a strategy for the identification and disruption of weakly oligomerized proteins. Structure. (Camb. ) 13, 7-15.

14. Wong,O.G.-W., Nitkunan,T., Oinuma,I., Zhou,C., Blanc,V., Brown,R.S.D., Bott,S.R.J., Nariculam,J., Box,G., Munson,P., Constantinou,J., Feneley,M.R., Klocker,H., Eccles,S.A., Negishi,M., Freeman,A., Masters,J.R., and Williamson,M. (2007). Plexin-B1 mutations in prostate cancer. Proc. Natl. Acad. Sci. U. S. A 104, 19040-19045.